Calcification Assay to Measure Calcium Concentration in Aortic Valve Interstitial Cells

Published: April 30, 2023

Abstract

Source: Bouchareb, R. et al., Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro.  J. Vis. Exp.  (2021).

This video demonstrates an assay technique for assessing the calcification of the aortic valve interstitial cells in vitro. The assay helps to quantify the calcium concentration using the arsenazo III reagent, a calcium-sensitive dye that reacts with calcium ions to form a purple-colored calcium-arsenazo complex.

Protocol

All animal procedures described here have been approved by Icahn School of Medicine at Mount Sinai institutional core and use committee. 1. Preparation before valve cell isolation from adult mice Clean and sterilize all the surgical instruments shown in Figure 1A by using 70% v/v ethanol and subsequently autoclaving them for 30 min. clean the surgical workspace with 70% ethanol. Add 500 µL of penicillin-streptomycin to 50 mL of 10 mm HEPES. P…

Representative Results

Figure 1: Description of valve dissection. (A) Representative image of all the surgical instruments needed for the dissection, scissors 2 is needed to open the skin of the mouse and scissors 3 to open the chest. Tweezers 5 and 6 are needed to hold the skin and open the chest. (B) Leave 3 mm of tissue from the aorta (black arrow). (C) Cut the hear…

Declarações

The authors have nothing to disclose.

Materials

3 mm cutting edge scissors F.S.T 15000-00
Arsenazo-III reagent set POINT SCIENTIFIC C7529-500 A kit to measure the concentration of calcium
Bonn Scissors F.S.T 14184-09
Calcium hydroxide SIGMA -Aldrich  31219
Collagenase type I (125 units/mg) Thermofisher Scientific 17018029
DMEM Tthermofisher 11965092
Extra fine graefe forceps F.S.T 11150-10
FBS Gibco  16000044
Fine forceps F.S.T Dumont
HCl SIGMA-ALDRICH H1758
HEPES 1 M solution STEMCELLS TECHNOLOGIES
L-Glutamine 100x Thermofisher Scientific A2916801
PBS 10x SIGMA-ALDRICH
Penicillin streptomycin 100x Thermofisher Scientific 10378016
Sodium Pyruvate 100 mM Thermofisher Scientific 11360070
Standard pattern forceps F.S.T 11000-12
Surgical Scissors – Sharp-Blunt F.S.T 14008-14
Trypsin 0.05% Thermofisher Scientific 25300054

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Calcification Assay to Measure Calcium Concentration in Aortic Valve Interstitial Cells. J. Vis. Exp. (Pending Publication), e21280, doi: (2023).

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